Throughout this application various publications are referenced by Arabic numerals. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosure of these publications in their entireties are hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains.
This invention relates to the detection and identification of protozoan and bacterial species, for example, the pathogenic bacterium Campylobacter pylori, by use of probes for 16S ribosomal RNA, and by use of the polymerase chain reaction (PCR).
Sensitive, rapid and reliable assays for the detection of specific microorganisms are very useful both for medical diagnostic purposes, and in the context of cell and tissue culture. Assays which involve culturing contaminating microorganisms can be time-consuming.
Assays based on the direct detection of species-specific DNA sequences in a sample using DNA probes avoid culturing, but can be hampered by the rarity of specific DNA sequences relative to the whole genome of a target microorganism and the consequent difficulty of obtaining sufficient amounts of specific DNA sequences for a successful assay. (1)
One means of overcoming these problems is amplification of DNA sequences by use of the polymerase chain reaction. The abundance of a specific DNA sequence can be increased for subsequent analysis by a factor of 10.sup.6 with this technique, which has been used for such purposes as the detection of abnormalities in human genes, DNA sequencing, and detection of viral pathogens (1).
Applicants' invention provides a means for detecting protozoan and bacterial species in a sample of, for example, culture medium, or in a tissue specimen, by use of particular primers for the polymerase chain reaction which are homologous to certain highly conserved sequences of DNA coding for the 16S subunit of ribosomal RNA. Bacterial species encode ribosomal RNAs which are functionally and evolutionarily conserved. The nucleotide sequences of bacterial 16S ribosomal RNAs diverge in regions termed "catalogs" (2). Bacterial 16S ribosomal RNAs possess three catalogs, connected by highly conserved "universal" sequences (3). Each catalog consists of approximately 400 nucleotides (25% of the 16S RNA). Phylogenetic classifications of species have been made by comparing "oligonucleotide catalogs" of bacterial 16S ribosomal RNAs. These regions provide targets for clinical detection and diagnoses based on molecular hybridization techniques (4). Lane (3) described these universal sequences, which were able to bind to 16S ribosomal RNA templates from over fifty microorganisms, both prokaryotic (such as bacteria and eukaryotic (such as protozoans). None of the references cited disclose the use of these universal 16S ribosomal RNA sequences as PCR primers for 16S ribosomal DNA catalogs.
Another means of overcoming the problems outlined above is to use probes that detect ribosomal RNA instead of DNA, because ribosomal RNA is abundant in the cytoplasm of all types of cells (71% of total cell RNA). Applicants' invention discloses a method for the production of species-specific DNA probes for the detection of protozoa or bacteria using PCR amplification of the species-specific sequences that exist between the binding sites of the universal 16S ribosomal RNA PCR primers; i.e., the "oligonucleotide catalogs" described above. This method may be also be used to produce species-specific PCR primers.
These assay methods provide rapid, sensitive, and efficient means of detecting bacterial or protozoan contamination of a sample or a specimen.
Campylobacter pylori is a bacterial strain thought to play an important causative role in chronic antral gastritis and gastric peptic ulcer diseases (5,6). C. pylori thrives on the surface of the stomach and produces ureases which neutralize hydrochloric acid. Diagnostic methods of detection of C. pylori, which could play an important role in the prevention and improvement of these disorders, would therefore be very significant.
Recent studies have revealed that the gastric mucosa of patients undergoing gastroduodenoscopy for upper abdominal complaints is often colonized by Campylobacter pylori. The presence of C. pylori has been closely associated with histologically proven gastritis and peptic ulcers (for review, see reference (7). Rauws (8) has shown a relationship between C. pylori infection and active chronic gastritis, and has demonstrated ultimate normalization of gastric mucosa after successful eradication of C. pylori. Since eradication of this organism from the gastric mucosa alleviates symptoms, early detection and treatment is important (9).
At present the urease test is the conventional method for detecting C. pylori, however false-positive and false-negative results are sometimes encountered (10, 11, 12, 13). To overcome these potential misdiagnoses, applicants disclose more accurate tests for the detection of C. pylori.
As discussed above, ribosomal RNA is a useful target for a nucleic acid probe because of its abundance. Gen-Probe produces kits for the detection of mycoplasma species (14), and for any member of the Legionellaceae family of bacteria (15) using cDNA probes for ribosomal RNA specific to mycoplasmas, or to Legionella species. However these kits do not detect specific bacterial species, in fact their purpose is not to detect specific species but to provide a broader range of detection.
Romaniuk (6) has sequenced unique, species-specific regions of Campylobacter pylori 16S ribosomal RNA. Using these sequences, applicants have developed a useful and practical DNA-RNA hybridization assay for the detection and identification of Campylobacter pylori in the gastric mucosa. This technique utilizes a [.sup.32 P]ddATP-labeled synthetic oligonucleotide probe complementary to nucleotide sequences present in C. pylori 16S rna. This probe is very sensitive and reacts with all 23 strains of C. pylori tested. It is also highly specific since there was no cross-reactivity with any of the following heterologous organisms: C. coli, C. fetus, C. jejuni, C. laridis and Escherichia coli. Hybridization of the oligonucleotide probe with C. pylori RNA was completely inhibited by treatment of the membrane filters with RNase but not by treatment with DNase. Although a gastric mucosa tissue homogenate slightly inhibited the hybridization, as few as 10.sup.4 C. pylori cells can be detected even in the presence of 5 mg of gastric mucosa.
Applicants have also developed a procedure for the detection of Campylobacter pylori in samples using the polymerase chain reaction. PCR amplification with universal ribosomal 16S gene primers was initially used to amplify a portion of the C. pylori 16S ribosomal gene. After DNA sequencing of the amplified fragment, primers specific for PCR amplification of the C. pylori 16S ribosomal gene were designed. With these primers, Campylobacter pylori can be uniquely detected, even when present in small quantities, by performing a polymerase chain reaction. There is no need to identify the DNA fragments resulting from the PCR amplification, because only Campylobacter pylori 16S ribosomal DNA will be amplified.
This PCR assay is even more sensitive than the RNA dot blot technique described supra. Furthermore, its ease and the fact that it does not require radioisotopes make this assay promising for clinical detection of C. pylori.
These two assays provide a rapid, sensitive, and efficient means for detecting Campylobacter pylori in gastric tissue specimens.